The project's goal consists in the development of novel diagnostic assay for use in clinical and field conditions. In this project we suggest a new DNA array-based technology for robust, isothermal, ultrasensitive colorimetric detection of pathogen-specific DNA sequences in test samples taken from potentially infected individuals. A novel strategy is based on the ability of peptide nucleic acid (PNA) to open up unique short sequences (20- 30bp), which makes it possible to detect DNA signature sequences within genomic DNA under non-denaturing conditions. Specific PNA-DNA construct will serve as exceedingly selective and very effective biomarkers. Ultra-sensitivity will be provided by two steps of signal amplification: each PNA-DNA construct triggers a polymerase-mediated rolling-circle amplification (RCA) to produce a linear array of DNA enzyme sensors; this DNAzymes catalyses chemical reactions with multiple turnover that generate chemiluminescence or produce colorimetric output. This synergistic combination a set of proven techniques is expected to overcome a long standing challenge: the detection of pathogenic microorganisms without target amplification and will yield an unconventional, non-PCR isothermal assay for expedient foolproof identification of biodefense and emerging pathogens not achievable by alternative techniques. The clear visual readouts and isothermal nature make this assay extremely suitable for application in resource-limited setting. The project relies on our proof-of- principle data, which demonstrates that we were able to detect about 20 bp-long, single-copy DNA signature sites in model bacterial genomes. During the project time, we plan to significantly extend our pilot data for pathogen identification based on the specific features of the genome (e.g. detect their pathogenic virulence factors and distinguish drug-resistant and drug-sensitive bacteria). The current requested funding will move technology toward applying it to clinical relevant cases and quickly advance the transition from a sensitive research tool to standard medical diagnostics. Once developed for pathogens under study, the combined workflow would be readily adaptable for rapid sensitive detection of other clinically relevant organisms.